We propose to employ computational protein modeling and aqueous-phase molecular dynamics methods to develop three-dimensional solution structural models based on partial X-ray crystal structural data and comparative homology modeling techniques. During the requested funding period, we propose to develop structural assembly of multi-domain proteins associated with intrinsic blood coagulation pathway. The specific objectives of the project are: (Aim I): to develop the dynamically equilibrated structural models for factors VIIIa, IXa and FX (Aim II) to model the binary complex between co-factor FVIIIa and factor FIXa (also known as intrinsic Xnase complex). (Aim III) to build the ternary complex among factors FVIIIa, FIXa and zymogen factor FX in an effort to delineate the protein recognition sites during FX activation. (Aim IV) to develop the binary complex between enzyme FIXa and zymogen FX to understand co-factor independent association of FIXa:FX By providing a structural understanding, we hope to address the following questions: i) What specific domains of the individual proteins, i.e., FVIIIa and FIXa, are involved in the intrinsic Xnase complex formation? Ii) How different are the domain-domain interactions and conformations upon co-factor binding? iii) What is the activation mechanism for zymogen FX proteolysis in intrinsic pathway? And iv) Can we decipher the structure-function correlation between the vast amount of hemophilia A and B patient mutational database and the proposed structural models, and if yes, can one use the protein- protein interaction data at the atomic details to engineer the therapeutic proteins? PHS 398/2590 (Rev. 09/04) Page 9 Continuation Format Page Principal Investigator/Program Director (Last, First, Middle): Venkateswarlu, Divi PROJECT NARRATIVE This research project, under NIH's Academic Research Enhancement Award (AREA) program, is to apply modern computational modeling and simulation methods to understand the dynamic nature of the protein- protein interactions associated with the intrinsic pathway of blood coagulation cascade. The complex process of coagulation involves a series of enzymatic reaction mechanisms that are auto-regulated by various activation-deactivation pathways. At least twenty proteins are known to play a major role in the cascade (Davie et al, 1991). Intrinsic pathway of blood coagulation involves the proteolytic activation of zymogenic factor X by serine protease IXa in the presence of phospholipids and divalent metal ions. Factor IXa is known to activate factor X at biologically significant rate only in association with co-factor VIIIa (Duffy & Lollar, 1992). The importance of co-factor FVIIIa-factor FIXa enzyme complex and its role in FX activation is illustrated by hemophilia in which the absence of either protein causes life-threatening bleeding disorders. Deficiency or defect in FVIIIa results in hemophilia-A which occur in 1 per 5000 patients. Currently, there is no cure for hemophilia and the existing therapy includes administration of recombinant FVIII or B-domain deleted rFVIII (Abshire et al, 2000). Similarly, genetic mutations in FIXa are associated with the bleeding diatheses hemophilia B. In contrast, high levels of FVIII activity have recently been reported to be associated with an increased risk of thrombosis (Kraaijenhagen et al, 2000). Thus, there is a great deal of interest in understanding the molecular structure of the three proteins VIIIa,IXa and FX and to develop a structure-function relationship among the three proteins and correlate with the known genetic mutations in FVIIIa (about 270 are known so far) with the structure. We plan to develop a complete molecular mapping of protein interaction sites among the human forms of enzyme (FIXa), substrate (FX) and co-factor (FVIIIa) proteins associated with the intrinsic pathway. We propose to employ computational protein modeling and aqueous-phase molecular dynamics methods to develop three-dimensional solution structural models based on partial X-ray crystal structural data and comparative homology modeling techniques. During the requested funding period, we propose to develop structural assembly of multi-domain proteins associated with intrinsic blood coagulation pathway. The specific objectives of the project are: (Aim I): to develop the dynamically equilibrated structural models for factors VIIIa, IXa and FX (Aim II) to model the binary complex between co-factor FVIIIa and factor FIXa (also known as intrinsic Xnase complex). (Aim III) to build the ternary complex among factors FVIIIa, FIXa and zymogen factor FX in an effort to delineate the protein recognition sites during FX activation. (Aim IV) to develop the binary complex between enzyme FIXa and zymogen FX to understand co-factor independent association of FIXa:FX (Aim V) to train graduate and undergraduate students in learning and applying modern computational protein modeling/bioinformatics methods to the problems in biochemical sciences. The training program is expected to provide a vehicle for the students at the Investigator's Institution, which is a predominantly minority serving HBCU, enter the biomedical research programs at research-intensive universities for doctoral and other higher studies. By providing a structural understanding, we hope to address the following questions: i) What specific domains of the individual proteins, i.e., FVIIIa and FIXa, are involved in the intrinsic Xnase complex formation? Ii) How different are the domain-domain interactions and conformations upon co-factor binding? iii) What is the activation mechanism for zymogen FX proteolysis in intrinsic pathway? And iv) Can we decipher the structure- function correlation between the vast amount of hemophilia A and B patient mutational database and the proposed structural models, and if yes, can one use the protein-protein interaction data at the atomic details to engineer the therapeutic proteins? [unreadable] [unreadable] [unreadable]